Kidney International, Vol. 39 (1991), pp. 1184—1192

Blood pressure and tubuloglomerular feedback mechanism in chronically salt-loaded spontaneously hypertensive rats YASUYUKI UsHI0GI, TOSHIKAZU TAKABATAKE, and DIETER ALBERT HABERLE Physiologisches Insrilut der Universitat München, Munich, Germany, and First Department of Internal Medicine, School of Medicine, Kanazawa University, Kanazawa, Japan

Blood pressure and tubuloglomerular feedback mechanism in chroni-

cally salt-loaded spontaneously hypertensive rats. Experiments were performed to qualitatively characterize the effects of tubuloglomerular feedback (TGF) inhibition by chronic salt loading on salt sensitivity of blood pressure in spontaneously hypertensive rats (SHR). After two weeks of salt loading, systolic blood pressure (SBP) was significantly exacerbated and plasma volume (PV) was expanded in salt-loaded SHR compared with those in control SHR (SBP: 182 1 vs. 159 2 mm Hg; PV: 4.38 0.06 vs. 4.04 0.03 ml/100 g body wt, respectively). Plasma volume of WKY was also but only transiently expanded by salt loading, whereas plasma volume expansion in SHR had persisted over the entire

dietary treatment period. TUF activity was assessed as the maximal reduction of single nephron GFR (SNGFR) on increasing loop of Henle perfusion rate from 0 to 40 nI/mm using previously collected tubular fluid from salt-loaded rats (TFs) or control rats (TFc). Maximal TGF response in salt-loaded SHR with TFs was 14.9 2.9% and 57.8 2.6% with TFc. In control SHR the responses were 16.9 2.5% with TFs and 52.7 2.9% with TFc. In salt-loaded WKY the response with TFs were

tubuloglomerular feedback (TGF) mechanism. This mechanism is generally believed to play a significant role in the regulation of

water and electrolyte balance as well as in the long-term regulation of the blood pressure. Three observations have led to

this idea. (i) The TGF mechanism influences the renal blood flow and glomerular filtration by changing the arteriolar flow

resistance at the glomerular vascular pole in response to changes of the luminal sodium chloride concentration at the macula densa and, in consequence, stabilizes the salt and water delivery to both proximal and distal nephron [91. (ii) Alterations of the activity of the TGF mechanism appear to influence the activity of the intrarenal renin-angiotensin system and, hence, the vascular tone of the arterioles of the parent glomerulus

[9—11]. (iii) The magnitude of the feedback response is modulated by specific mechanisms under conditions of altered extra3.1 1.6% and 37.4 2.8% with TFc. And in control WKY, the cellular flui4 volume [9] and sodium intake. In particular, when 1.9% and 40.8 2.8% with TFc, response with TF5 were 8.2 respectively. These results indicate the TGF resetting in chronically the dietary sodium load is increased, the feedback response is salt-loaded SHR and WKY is caused by the activation of humoral TGF attenuated and contributes to the increase of sodium excretion inhibitory factor. The suppression of TGF in SHR was, however, far [12—18] by several distinct mechanisms. In rats chronically fed more variable and, on average, less than in WKY. In addition, the a high salt diet, the resetting of feedback system is caused by maximum TGF response elicited by TFs in SHR and systolic blood the activation of an inhibitory factor in tubular fluid [19] rather pressure in conscious donor SHR were significantly correlated (r = than by a change in the intrinsic characteristics of the juxtaglo0.8142), This indicates that in salt-loaded SHR less activation of the TGF inhibitory factor goes in hand with a greater blood pressure merular apparatus, as is the case in rats treated chronically with increase. It is thus reasonable to propose that the renal response to DOCA and isotonic saline for drinking [201. In contrast, this chronic salt-loading in SHR might be blunted by defective activation of the feedback inhibitory substance. This defect may contribute to the exacerbation and maintenance of hypertension in this strain.

A number of studies [1—51 have demonstrated that chronic dietary salt loading exacerbates the development and severity of hypertension in spontaneously hypertensive rats (SHR). Since previous studies have identified various functional abnormalities in the kidneys of these rats [6, 7] and since the renal function is involved in long-term blood pressure regulation [81, it is possible that additional, unknown abnormalities of the renal sodium balancing mechanisms participate in the exacerbation of hypertension during chronic volume expansion. One mechanism of particular interest in this respect is the Received for publication August 27, 1990 and in revised form January 24, 1991 Accepted for publication January 25, 1991

© 1991 by the International Society of Nephrology

inhibitory principle is not detectable in tubular fluid of rats acutely volume expanded by the infusion with Ringer's solution and appropriate amounts of fresh rat plasma [21]. Since the TGF function is reportedly disturbed in prehypertensive SHR [221, we investigated the activation and the effectiveness of the humoral inhibitory factor in SHR and WKY and

its relationship to the increase of blood pressure in chronic dietary salt loading. An inverse correlation between the blood pressure increase and the activity of the inhibitory factor was found which may indicate an abnormal response of the SHR kidney and/or its hormonal regulating systems to dietary salt loading. Methods

Dietary treatment Experiments were performed on male 6- to 7-week-old spontaneously hypertensive rats (SHR) and age-matched normotensive Wistar-Kyoto rats (WKY) (Savo-Ivanovas, Kisslegg, Germany). The rats were divided into two groups. One group was fed a high salt diet (Altromin C 1036, containing either 40 or 100

1184

Ushiogi et a!: TGF resetting in SHR

g NaCI/kg food) for at least two weeks. The other group was fed a low salt control diet (Altromin C 1000, containing 0.35% Na,

1185

similarly (1:10) and optical density determined in a dual beam

spectrophotometer (model MPS 2000, Shimadzu) at wave 0.48% Cl and 0.23% K). Food and water were available ad lengths of 740, 660 and 610 nm. The difference in the optical libitum throughout the study and the consumption of both was densities at 660 and 610 nm was corrected by subtracting the checked randomly. Systolic blood pressure and pulse rate in optical density at 740 nm and plotted against Evan's Blue conscious, prewarmed rats were measured by the tail cuff concentration. method once weekly and twice in the three to five days prior to

Collection of tubular fluid Late proximal tubular fluid (TF) was harvested under suction pressure. Body weight was determined on the same day as the at a rate of 8 to 10 nl/min into an oil-filled siliconized micropipette (outer tip diameter 8 to 10 tm), the tip of which had been blood pressure measurement. platinum glazed to make it visible, and which was mounted in a Animal preparation microperfusion/suction pump (Walter Klotz, Munich, GermaMicropuncture experiments were performed two to four ny). Endogenous tubular fluid was collected from the experi-

the micropuncture experiment. The average of four or five replicate measurements was used as an estimate of blood

weeks after beginning dietary treatment. Rats weighing be- mental rat. Exogenous tubular fluid was collected from a rat tween 190 and 270 g were anesthetized by intraperitoneal from the other dietary group prepared in parallel. injection of mactin, 100 mg/kg body weight (Byk-Gulden, Feedback response Konstanz, Germany). The rat was placed on a thermostaticallycontrolled heated table to maintain rectal temperature at apThe micropuncture protocol for estimating tubuloglomerular feedback response was as follows: The course of a nephron was and vein were catheterized. A solution of polyfructosan (125 identified by injecting small volumes of Ringer's solution cong/liter in 150 mmollliter NaCI) was infused at a rate of 4 ml/hr/kg taining FD & C green (2 g/liter) into a randomly-selected body wt in control group and 5 mI/hr/kg body wt in the proximal tubular loop. The harvested tubular fluid or Ringer's salt-loaded group. The arterial catheter was used to monitor solution was then perfused into the ioop of Henle at a rate of 0, blood pressure and to withdraw blood samples. The left kidney 10, 20 or 40 nl/min from the microperfusion pipette inserted into was then exposed through a left subcostal flank incision and the last accessible proximal loop. Usually one Ringer's perfusupported in a plastic cup using oil-soaked cotton wool. The sion and two to three tubular fluid perfusions were performed kidney was immobilized in agar (30 g/liter in 150 mmollliter per rat in random order. After an equilibration period of about NaC1) and the renal surface bathed with warmed (38°C) mineral two minutes, a micropipette (outer diameter 10 sm) filled with Sudan Black-stained paraffin oil was inserted into the earliest oil. The left ureter was catheterized for urine collection. proximately 37.5°C. After tracheotomy, the right femoral artery

Measurement of GFR and plasma volume Rats were prepared as described above. After an equilibration period of approximately 60 minutes, a timed urine collection was begun for the determination of glomerular filtration rate and electrolyte excretion. Arterial blood samples were

taken before and after urine collection. Serum and urinary polyfructosan concentrations were measured by perchioric acid

hydrolysis to fructose and determination of the latter by the hexokinase/G-6-P dehydrogenase method, using a commercially available kit (Glucose/fructose Kit No. 139 106, Boehringer Mannheim, Mannheim, Germany). GFR (polyfructosan clearance) was determined using the standard formula. Serum and urinary sodium and potassium concentrations were determined by flame photometry. Chloride concentrations were determined by the Cotlove method (Chloridometer Buehler Instruments, Fort Lee, New Jersey, USA). Plasma volume

accessible proximal segment, an oil block injected, and a timed, quantitative collection of tubular fluid begun for the determination of single nephron glomerular filtration rate (SNGFR). After completion of the sample, the perfusion rate was changed and further collections made. Each subsequent collection was made slightly upstream to avoid the possibility of leakage. The volumes of tubular fluid were measured by injection into

a constant-bore microcapillary (Microcap 0.5 tl, Drummond) and measuring the length of the column by means of an ocular micrometer. Inulin in tubular fluid was determined by a microadaptation of the same method used for urine and plasma samples. SNGFRs were calculated using the standard clearance expressions and expressed in nl/min/g kidney weight. NaCI loading Another series of experiments were performed to examine the effect of more effective salt loading and possibly more remarkable volume expansion. Some SHR were fed 100 g/kg NaC1 diet (Altromin C 1036, 100 g NaCl/kg dry food) during at

In a separate series of experiments, plasma volume was least two weeks before the experiments. The systolic blood measured by the Evans blue dilution technique at two, three pressure and plasma volume were measured only at two weeks and four weeks after beginning dietary treatment. After prepa- of salt loading. Micropuncture experiments were performed in

ration as above and following an equilibration period of 30 the same manner as above, but the determinations of SNGFR minutes, a 500 zJ blood sample was taken and subsequently were made only by loop perfusion with endogenous TF. used to prepare the standard concentrations of Evans Blue. Statistics After further 10 minutes, 100 p! of a 100 mgldl solution of Evans Results are given as means SEM. Data were tested using Blue (E. Merck, Darmstadt, Germany) in Ringer's solution was injected i.v. as a bolus. A period of 10 minutes was allowed for

analysis of variance and linear regression analysis. The signif-

distribution, then a second sample taken and the Evans Blue concentration measured. Plasma and standards were diluted

icance of differences in means was established by Tukey's method for multiple comparisons [231 and by the Kruskal Wallis

Ushiogi et al: TGF resetting in SHR

1186

* were slightly but significantly expanded in both salt-loaded

200

WKY and SHR (4.52 0,05 and 4.38 0.06 ml/l00 g body wt, respectively, compared with 4.12 0.04 and 4.04 0.03 ml/lOO

E

a aa a

g body wt in controls). Plasma volume expansion was still demonstrable at four weeks in salt-loaded SHR (4.37 0.05 compared with 3.91 0.03 ml/lOO g body wt). In contrast,

*

180

160

plasma volume in salt-loaded WKY decreased with increasing length of dietary treatment and was no longer different from that in controls at four weeks (4.08 0.05 compared with 4.04

———

0.07 mlIlOO g body wt).

Clearance results blood pressure and renal clearances obtained during micropuncture experiments are presented in Table 2. Mean blood pressure was significantly higher in salt loaded SHR than in control SHR. There were no differences in urine volume and whole kidney GFR between salt-loaded and control groups within each strain. Urinary sodium excretion in salt-loaded groups was significantly greater than in controls. Urinary chloride excretion was increased after salt loading although the increases in WKY failed to reach significance. With the exception of mean blood pressure there was no difference between the two strains in any parameter measured.

0 0 0 0 0 0 a >-

Mean

140

U)

120

100

I

L

—

Feedback analysis

Figure 3 and Table 3 demonstrate the response of SNGFR to loop of Henle perfusion in SHR. Three perfusates were used: Fig. 1. Effect of dietary salt loading on systolic blood pressure mea- endogenous TF, exogenous TF (from a rat of the other dietary sured by tail-cL4ff method in salt-loaded and control SHR and WKY. group), or Ringer's solution. In control SHR, loop of Henle SCM. * Indicates significant differences perfusion with endogenous TF elicited a significant decrease of Values are given as means from systolic pressure in control SHR. Symbols are: (——) salt-loaded SNGFR on increasing perfusion rate from 0 to 20 or 40 nl/min. SHR; (--0--) control SHR; (—A—) salt-loaded WKY; (--Li--) control In contrast, the feedback response was significantly attenuated WKY. when the loop of Henle was perfused with exogenous (high salt) TF. The response of SNGFR to loop perfusion with Ringer's solution was similar to that with endogenous TF. Test (H-test). A probability level of less than 0.05 was considThe feedback response in salt-loaded SHR was significantly ered significant. attenuated when the loop of Henle was perfused with endogenous TF, whereas much greater feedback responses were Results induced with exogenous TF or Ringer's solution. The feedback Blood pressure, pulse rate and plasma volume responses to any given perfusate were qualitatively similar in There was no difference in blood pressure between the two both control and salt-loaded SHR whereby the response to the groups of SHR before the dietary pretreatment. After one week high salt SHR TF was considerably less uniform than that to on the diets, systolic blood pressure in salt-loaded SHR was high salt WKY TF. Figure 4 and Table 3 show the feedback response of SNGFR significantly higher than in control SHR. Systolic blood pressure in both groups of SHR continued to rise over four weeks of in WKY. Again, in both salt-loaded and control WKY, the salt loading, but systolic blood pressure in salt-loaded SHR feedback response was significantly attenuated when the loop remained significantly higher than that in control SHR (Fig. 1). of Henle was perfused with high salt TF, whereas perfusion In salt-loaded conscious SHR, systolic blood pressure was with control TF elicited practically normal feedback responses. higher than the mean blood pressure in anaesthetized rats. Both The characteristics of the tubuloglomerular feedback function blood pressures were significantly correlated (r = 0.5957). The in WKY were, however, different from those in SHR. Maxilinear regression line is given by BPmeananesth = 25.5 + 0.696 mum stimulation of the TGF by loop perfusion at 40 nllmin with BP8 (N = 54). Systolic blood pressure in WKY showed no Ringer's solution in control SHR depressed SNGFR signifisignificant response to salt loading. There was also no differ- cantly more than in WKY (53.0 3.2% vs. 35.6 1.4%; Fig. 5). ence in pulse rate in the salt loaded and control groups of both This may imply that TGF in normal SHR is hyperactive strains. Food intake was comparable in all groups. Body compared with WKY. The feedback inhibitory activity was significantly different weights of WKY were significantly greater than those of SHR, but high salt diet had no effect on body weight in either SHR or between the two strains on salt loading. Firstly, the maximal feedback response in salt loaded SHR was significantly higher WKY (Table 1). The specific effects of dietary salt-loading on plasma volume than in WKY rats (Table 3, compare Fig. 7). Interestingly, the are presented in Figure 2. After two weeks, plasma volumes variance in salt loaded SHR was significantly larger than in salt 0

1

2

Time, weeks on diet

3

4

1187

Ushiogi et a!: TGF resetting in SHR

Table 1. Effects of two weeks dietary salt loading on systolic blood pressure (SBP), heart rate (HR) in SHR and WKY, and body weight (Weight)

SBP mm Hg SHR WKY

182 119

1

(29)

High salt diet HR

Weight

beats/mm

g

354

5 (29)

362 4 (20)

2 (20)''

211

237

SBP mm Hg

159 2 (28)

3 (29) 2 (20)"

119 2 (20)"

Control diet HR

Weight

g

beats/mm

358 354

4 (28) 6 (20)

207 234

2 (28) 2 (20)"

Data are shown as means SEM with number of experiments in parentheses. a Significant difference from the corresponding control value b Significant differences between SHR and WKY

the ratios, which takes into account the difference of the intrinsic sensitivity of TGF in SHR and WKY, was also

SHR 5.0

significant. The attenuation of TGF in salt-loaded SHR was, therefore, less pronounced than in salt-loaded WKY. There was 4.5

4.0

-c

0

I

II

F

II

IHHI

WKY

E

5.0

0 >

no difference in the ratio between two control groups of rats, 1.03 0.08 in control SHR, 1.16 0.10 in control WKY. Figure 6 shows the relationship between maximum feedback response (% reduction in SNGFR) elicited by TF harvested in a saltloaded SHR and the previously measured conscious systolic blood pressure in these donor rats. A comparable relationship exists between the TGF response and the mean arterial blood pressure of the same donor rat during anaesthesia. This relationship is described by the equation (% reduction of SNGFR) = 0.923 (mean arterial blood pressure) — 135 (r = 0.65, P = 0.0014). Thus, it may be concluded that there is an inverse, highly significant correlation between the maximum feedback response and the arterial blood pressure of the donor rat. Effect of 100 glkg NaCI diet After two weeks of salt loading, systolic blood pressure in SHR fed a 100 g/kg NaCI diet was significantly higher than in SHR fed a 40 g/kg NaC1 diet (198 4 mm Hg, N = 10, vs. 182 1 mm Hg, N = 29). Plasma volume in the 100 g/kg SHR was 4.75 0.14 mlIlOO g body wt, not significantly different from that in the 40 g/kg NaCl SHR group. SNGFR during loop

Co

E

Co Co

0

perfusion with endogenous TF at rates of 0, 10, 20 and 40 ni/mm

'1' r

11111111

1

HH1I

I

Time, weeks on diet

Fig. 2. Effects of dietary salt loading on plasma volume in SHR and WKY. Symbols are: (Li) control rat; (11111) salt-loaded rat. Data are means SEM. The numbers of observations for the panels in the upper part of

the figure were 8, 7, 9, and 9, respectively, and in the lower part, 7, 7, 5, 6, 8, and 7, respectively. * Indicates significant differences between salt-loaded and control rats. • indicates a significant difference between salt-loaded SHR and WKY at four weeks.

were 35.9 1.9 (N = 16), 33.4 1.4 (N = 4), 30.8 3.4 (N = 2.6 (N = 12) ni/min/g kidney wt, respectively. 8) and 23.0 Maximal reduction of SNGFR on increasing the perfusion rate from 0 to 40 nl/min was 12.8 1.1 nllmin/g kidney wt, which represents a 36,8 3.1% reduction (Fig. 7). This is significantly higher than in the 40 g/kg NaC1 SHR group. The response of SNGFR to loop perfusion was not as uniform as in salt-loaded or control WKY or control SHR. Discussion

Different effects of salt loading in SHR and WKY

In the present study there were three major differences between WKY and SHR in the response to chronic dietary salt

loading. Firstly, consistent with previous reports [1, 2, 5J, systolic blood pressure increased more rapidly and to higher loaded WKY. Secondly, the ratio of the maximum reduction of levels in salt-loaded SHR than in controls. WKY showed no SNGFR on ioop perfusion with endogenous tubular fluid to that with Ringer's solution in the same rat was in salt-loaded SHR, 0.33 0.07 (18 pairs of tubules, 8 rats) and in salt-loaded WKY, 0.05 0.04 (10 pairs of tubules, 6 rats). The difference between

response of blood pressure to salt loading. Secondly, after two

weeks of salt loading plasma volume in both strains had increased to a comparable extent. However, plasma volume in WKY then declined progressively and returned to normal level

Ushiogi et a!: TGF resetting in SHR

1188

Table 2. Summary of systemic and renal function in the micropunctured kidney in experimental animals WKY

SHR MBP mm Hg

Control

Salt loaded

2 (19)

165 21

152

1.06

GFR mi/minig kidney wt

(21)

1.10

0.03 (17) 4.00 0.17 (20) 572.6 93.P' (14) 817.0 39.3 (14) 805.6 83.5a (13) 0.277 o.ossa (9)

0.03

(17)

.

UV td/minIg kidney wt

3.59

0.17

(17)

VUNa nmol/minlg kidney wt .

VU nmol/min/g kidney wt

6.9

38.8

(13)

67.6

864.5

(13)

.

VU1 nmol/min/g kidney wt

60.2

346.8

(11)

0.005

0.022

FENa %

(9)

Control 104

2b

(14) 1.07 0.01 (10) 3.61 0.31

(II)

7.7 (7) 845.9 150.4 (7) 401.6 56.6 (6) 0.034 0.005 (6) 54.5

Salt loaded 103

(24) 0.02 (15) 3.97 0,17 (14) 465.4 67.5a (10) 617.0 61.4 (10) 703.0 89.2 (10)

1.10

0.319 o.o4o (9)

Abbreviations are: MBP, mean blood pressure; GFR, glomerular filtration rate; Us', urine volume; 'UNa VUK, VU1, urinary excretion of sodium, potassium and chloride, respectively; FENa, fractional excretion of sodium. Data are given as means SEM, with number of experiments in parentheses. a Significant differences between salt-loaded and control rats b Significant differences between the two strains

Control SHR

Salt-loaded SHR

Q)

40

40 -

30 -

30 -

20

20 -

E

Fig. 3. Relationship between SNGFR measured in proximal tubule and loop of Henle perfusion rate in salt-loaded and control SHR. Symbols are: (—•—) endogenous tubular fluid; (--0--) exogenous tubular

I1)

fluid; (—A—) Ringer's solution. Data are given as means SEM. * Indicates a significant difference 10 4

10

J..

10

20

from the SNGFR at the same perfusion rate with exogenous tubular fluid. ** Indicates a significant different from SNGFR with exogenous tubular fluid or Ringer's solution. ++ indicates a significant difference from SNGFR with endogenous tubular

Perfusion

rate, n//rn/n

fluid or Ringer's solution.

4

J.

0 :i- '

0 0

10

20

Perfusion rate, n//rn/n

40

0

after four weeks. In contrast, the plasma volume expansion in SHR persisted over the entire dietary treatment period. These results are consistent with previous reports on SHR [24] or Dahl-S rats [25], and with recent observations on MunichWistar rats in which plasma volume expansion on salt loading

TGF activity is assayed by loop of Henle perfusion with

observations from this laboratory [13, 19] and elsewhere 118]. This attenuation is, however, less pronounced in SHR than in WKY. Three observations lead to this conclusion: (i) When

Consistent with an earlier report [19], TGF resetting was caused

Ringer's solution, the dependency of SNGFR on the loop of Henle perfusion rate and, in particular, the response of SNGFR

to a supramaximal challenge (loop of Henle perfusion at 40 ni/mm) was similar in normal and salt-loaded WKY and in was also transient and in which the high salt intake was normal and salt-loaded SHR. However, in agreement with balanced without volume expansion (unpublished observations earlier reports [22, 26—291 both the amplitude of TGF curve, D.A. Haberle, V. Gross, C. Mast and C. Metz). Third, chronic that is, the maximum response, and TGF sensitivity (the slope salt loading markedly attenuated the feedback response in both of the curve at the turning point) was greater in SHR than in SHR and WKY due to the appearance of an inhibitory factor in WKY. Thus, in SHR TGF appears to be hyperactive and this the tubular fluid. This finding is consistent with previous hyperactivity appeared not to be affected by salt loading. (ii) by the activation of a humoral inhibitory principle in tubular fluid. This is concluded from the following: First, the feedback

1189

Ushiogi et a!: TGF resetting in SHR

Table 3. Single nephron GFR (SNGFR) values and maximal response of SNGFR (SNGFR max) in salt-loaded SHR (SHR salt), in control SHR (SHR cont), in salt-loaded WKY (WKY salt) or in control WKY (WKY cont) rats

LSNGFR max

SNGFR ni/rn in/g kidney wt

0

20

40

34.7 2.3 (10) 35.0 2.5 (5) 31.1 3.0 (3)

33.4 1.7 (21) 24.9 1.7 (9) 25.2 2.2 (6)

1.3' 28.3 (21)

38.6 1.7 (14) 38.1 1.8 (21) 39.2 2.3 (10)

36.7

27.8

34.5 1.4 (22) 36.1 1.9 (12) 35.2 3.4 (10)

34.6 1.8 (12) 33.5 2.3 (7) 34.3 3.5 (5)

41.2 2.2 (18) 41.3 1.8 (15) 41.7 1.3 (9)

42.3

SHR salt

TF end TF exo Ringer

SHR cont TF end TF exo Ringer

WKY salt TF end

TF exo Ringer

WKY cont TF end

TF exo Ringer

%

10

35.5

1.5

(30)

36.9

2.8 (10) 36.3 2.0 (9)

37.2

2.0 (9) (8)

40.9

2.7 3.0

36.8 2.8 (12) 28.3

2.6 (8) 1.8

(8)

0.4

39.1

3.0

(5)

(5)

40.5

1.9 (9)

(3)

33.5

3.2

(8) 30.2

1.9

(7) 26.2

1.7

(7) 31.8 2.0 (10) 36.7 2.8 (8) 32.0 0.6 (4)

15.6

1.3

(7) 17.3

1.5

14.9

29b,d (21)

2.6 (7) 52.9 2.4 57.8

(9)

(9)

2.9

1.3 (11) 31.5 2.0C (15) 18.7 1.9 (8)

52.7

31.5 0.8" (12) 22.5 1.8 (10) 22.3 2.1 (8)

3.1

(12) 37.4 2.8 (10) 39.3 1.9 (8)

23.0

1.7

40.8

l.4C

8.2

18.6

(11)

37.3 (9) 26.8

1.1

(9)

(11)

16.9

2.5C

(15)

53.0

3.2C

(8)

16"

2.8 (11) 1.9C

(9) 35.6 1.4 (9)

Perfusates are endogenous tubular fluid (TF end), exogenous tubular fluid (TF exo) or Ringer's solution (Ringer). Data are given as means SEM. a Significant difference from SNGFR at the same perfusion rate with exogenous tubular fluid b Significant difference from SNGFR and maximal response of SNGFR with exogenous tubular fluid or Ringer's solution Significant difference from SNGFR and maximal response of SNGFR with endogenous tubular fluid d Significant difference from maximal response of SNGFR in salt-loaded WKY on ioop perfusion with endogenous tubular fluid C Significant difference from maximal response of SNGFR in control WKY on ioop perfusion with Ringer's solution

activity in donor or normal SHR was less attenuated than with tubular fluid from salt-loaded WKY. This difference is even from high salt rats. Second, feedback responses in both salt more evident when feedback response to the test challenge with loaded and control rats of each strain were similar and "nor- Ringer's solution in salt loaded rats of both strains was taken mal" on loop perfusion with TF from control rats. Third, the into account. When the ratios between maximum reduction of TGF response in nephrons microperfused with TF from control SNGFR by loop perfusion with endogenous TF to that with rats was similar to that seen with Ringer's perfusion. Thus, Ringer's solution from each rat were compared between the since the TGF responses to the two types of tubular fluid differ strains, the ratio in salt-loaded SHR was significantly greater in the same animal, the fluids must differ in their properties. than that in salt-loaded WKY. There was no difference, howSince the TGF response to Ringer's perfusion was similar to ever, between the ratio in control SHR and control WKY. This that with TF from control rats, the inhibition of TGF seen with difference in the salt loaded rats appears to be due to a less TF from high salt rats must result from the appearance of an homogeneous generation of the TGF inhibitory factor in SHR inhibitory principle in this tubular fluid. It is unlikely that the compared with WKY. Whereas salt loading in WKY resulted in inhibitory principle simply causes a decrease in the intraluminal similar TGF attenuation in all rats, the TGF in some SHR NaCl concentration at the macula densa segment. In compara- appeared not to be attenuated at all. ble experiments on salt-loaded Munich-Wistar rats NaCI delivDefective TGF inhibition and blood pressure ery to the early distal tubular segment was similar in salt-loaded and control rats [131. Moreover, in both strains of rats dietary Interestingly, the increase of blood pressure on salt loading in salt loading caused a significant increase of urinary sodium and the latter rats was greater than in SHR in which the TGF was chloride excretion compared with control. The nature and the attenuated to a similar extent as in WKY. This conclusion is mechanism responsible for activation of the feedback inhibitory based on the following observations: Firstly, systolic blood factor are not clear and require further elucidation. (iii) As pressure of the (conscious) salt-loaded donor SHR is signifimentioned above, with TF from salt-loaded SHR the feedback cantly correlated to the maximum feedback response elicited by response in both salt-loaded and control rats of each strain was markedly and similarly attenuated on loop perfusion with TF

Ushiogi et a!: TGF resetting in SHR

1190

Control WKY

Salt-loaded WKY

40 -

40

30 -

30

-I m IL.

20 -

Fig. 4. Relationship between SNGFR measured

20

in proximal tubule and loop of Henle perfusion rate in salt-loaded and control WKY. Data are given as means SEM, Symbols are: (—I—) endogenous tubular fluid; (--0--) exogenous

U)

tubular fluid; (—A—) Ringer's solution. ** Indicates a significant difference from SNGFR at

10

0

10

20

Perfusion rate, ni/rn/n

-,

01,

40

0

10

40

20

Perfusion rate, n//rn/n

a same perfusion rate with exogenous tubular fluid or Ringer's solution. + indicates a significant difference from SNGFR with exogenous tubular fluid or Ringer's solution.

its TF in control or salt-loaded SHR. No such correlation was seen in control SHR. Two observations exclude the possibility that this correlation results from a different pressure natriuretic response within the group and hence a relative difference of sodium loading. First, there was no negative correlation between plasma volume and systolic blood pressure in salt-loaded

Stress in conscious SHR increases blood pressure and renal sympathetic nerve activity and decreases urinary sodium excretion [35]. This antinatriuretic effect of stress in SHR is enhanced by chronic sodium loading, whereas similar stress in

Defective TGF inhibition and volume expansion

retention and to return the expanded plasma volume to normal.

conscious WKY has no effect on sodium excretion [361. Finally,

as discussed above, without the inhibitor in tubular fluid the SHR. Second, when salt loading of SHR was increased by tubuloglomerular feedback system is hyperactive in salt-loaded feeding a 100 g/kg NaC1 diet a similar relationship between and normal SHR compared with WKY rats, also possibly a systolic blood pressure and feedback activation with tubular consequence of increased sympathetic nerve activity (see fluid was observed. This suggests that the defective activation above). This follows from earlier observations which demonof the feedback inhibitory substance and the sensitivity of the strated a marked attenuation of this hyperactivity after renal blood pressure to salt loading in SHR might be in some way denervation. Similar denervation in WKY did not influence the causally interrelated. The underlying mechanisms are not un- feedback activity [281. It might thus be argued that firstly, in salt-loaded SHR, TGF inhibition, had it been present, would derstood at present. not have been adequate to overcome the increased sodium

It is not clear whether or not the persistence in plasma Secondly, since these SHR rats have a primary increase in volume expansion is due to the defective feedback resetting in salt-loaded SHR rats. That plasma volume correlates neither with the level of systolic blood pressure (and nor, presumably, with the level of TGF activity) in salt-loaded SHR and that TGF resetting and plasma volume do not correlate in salt-loaded WKY might be regarded as evidence against such a relationship. However, in view of the other sodium retaining mechanisms activated in SHR and their coexistence with pressure diuresis, the above conclusion may be premature. Salt loading in SHR, for instance, has no effect on plasma atrial natriuretic factor (ANF) concentration [30]. In addition, the capacity for

blood pressure, the increased sodium retention will be partly balanced by enhanced pressure natriuresis resulting—as observed—in comparable urinary excretion of electrolytes during salt loading in both SHR and WKY. These mechanisms may compensate to some extent the defective TGF inhibition, resulting in virtual independency between plasma volume and TGF activity.

impaired in SHR compared with WKY [311. Moreover, sympathetic nerve activity, which has been shown to stimulate tubular salt and water reabsorption in both anaesthetized and conscious animals [32—34], is significantly increased in SHR and further enhanced by sodium loading. The pathophysiological relevance

TGF by stimulating the production of a vasodilating and natri-

of this mechanism is apparent in the following observations:

inhibitor produced during chronic salt loading. In addition,

Unjfying hypothesis A unifying, but admittedly completely unproven, hypothesis

might be advanced with respect to these considerations. If it synthesis of vasodilatory prostaglandins on salt loading is were assumed that the TGF inhibitor in tubular fluid inhibits

uretic substance in the JGA, this substance might not only prevent the vasomotor effect of a stimulation of the macula densa by a high luminal NaCI concentrations but also compensate the blood pressure raising effect of putative Na-K-ATPase

1191

Ushiogi et al: TGF resetting in SHR

50

0—

S 40

0

$ U-

(9

z(I)

10 — a)

30

0

0 0

0

8

U

•0

-U a)

E 0

20

0

S

U-

(9

z(I)

0

U a) (a (a a)

U

0

10 —

20 —

0a)

8

S

0-

I 190

180

170

200

Systolic blood pressure, mm Hg

30

I

I

0

10

I 20

40

Fig. 6. Relationship between the maximal response of SNGFR in salt-loaded (•) and control (0) SHR on loop perfusion with tubular fluid from salt-loaded SHR and the systolic blood pressure of the conscious donor SHR. r = 0.8142; P < 0.001.

Perfusion rate, ni/rn/n

1.2 r

Fig. 5. Relationship between the decrease of SNGFR and loop of

1.1

Henle perfusion rate with Ringer's solution in control SHR (-0-) and control WKY (-Lx-). Data are given as means SEM. * Indicates a significant difference from the corresponding value in control SHR.

1.0 U-

0.9

S S

z(I)

0.8

S

i-U

0.7

(9

because of its TGF inhibiting effects, this substance would contribute to increased sodium chloride excretion and hence, under chronic conditions and in concert with other mechanisms, enable the organism to excrete an increased sodium chloride intake without major expansion of the plasma and

U-

zU)

that the renal response to chronic salt loading in SHR might be

0.6

I

0.5

extracellular volume. In summary, tubuloglomerular feedback resetting in chronically salt-loaded SHR and WKY rats is caused by the activation of humoral feedback inhibitory substance. However, the activity of this factor in tubular fluid from SHR was not uniform and,

on average, less than in WKY. The maximum feedback response elicited by loop perfusion with tubular fluid from saltloaded SHR and the systolic blood pressure in the conscious donor rat were significantly correlated. Thus, in salt-loaded SHR less activation of the feedback inhibitory substance goes in hand with a greater blood pressure increase. In addition, plasma volume expansion by salt loading persists in SHR but was only transient in WKY. It is thus reasonable to propose

S

(9

0.4

SHR4g%

SHR1O9%

WKY4g%

Fig. 7. Maximal feedback responses by loop perfusion with endogenous tubular fluid in 40 g/kg NaCI diet-loaded SHR (SHR 4 g%) and WKY (WKY 4 g%), and 100 g/kg NaG! loaded SHR (SHR 10 g%). *

Indicates a significant difference from the value in 40 g/kg NaCl loaded Indicates a significant difference from the value in 40 g/kg

WKY. •

NaCI loaded SHR.

blunted by defective activation of the feedback inhibitory substance. This defect may contribute to the exacerbation and maintenance of hypertension in this strain.

1192

Ushiogi et at: TGF resetting in SHR

Acknowledgments The authors gratefully acknowledge the support of Professor Dr. Klaus Thurau as well as financial support of the Alexander v. Humboldt Foundation. We also thank Dr. J. Davis for his help with the manuscript and Ms. M. Stachl for technical assistance.

tubuloglomerular feedback responses to plasma expansion in the rat. Am J Physiol 235:F156—F162, 1978 18. SCHNERMANN J, SCHUBERT G, BRIGGS JP: Tubuloglomerular feed-

back responses with native and artificial tubular fluid. Am J Physiol 250:F16—F22, 1986

19. HABERLE DA, DAVIS JM: Resetting of tubuloglomerular feedback:

Evidence for a humoral factor in tubular fluid. Am J Physiol 246

Reprint requests to Yasuyuki Ushiogi, The First Department of

Internal Medicine, School of Medicine, Kanazawa University, 13-I Takaramachi, Kanazawa City, Ishikawa, 920 Japan.

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